The proposed work seeks to (1) provide biochemical evidence, and a genomic localization, for DNA sequence amplification in antifolate-resistant Chinese hamster cells which overproduce the enzyme dihydrofolate reductase, DHFR; (2) demonstrate identity between the putative DNA amplification event and HSRs (homogeneously staining regions) revealed by G-banding of metaphase chromosomes; and (3) determine the mechanism of overproduction of DHFR in these cells. With the use of combined cytological, molecular biological, and recombinant DNA techniques, which will include sister chromatid exchange analysis, mRNA purification, sequencing, cDNA synthesis via reverse transcription, in vitro labeling of DNA using "nick translation," Cot and Rot sequence complexity analysis, and cytological hybridization in situ, we will also attempt to provide evidence for structural gene amplification as a previously unrecognized mode of gene regulation in mammalian cells.